Enterohemorrhagic Escherichia coli (EHEC) are food-borne bacterial pathogens that cause severe and occasionally fatal hemorrhagic colitis (HC). Young children are disproportionately affected by severe manifestations of disease, and may develop hemolytic-uremic syndrome (HUS), a rapidly progressive and sometimes fatal form of renal failure. HUS is an uncommon complication of disease due to EHEC, but its importance is amplified by its severity, tendency to affect young children, and the absence of any specific treatment or preventative measure. EHEC is listed as a CDC/NIAID category B biohazard agent. When HUS does occur, it is often after recovery from the diarrheic phase. Intensive fluid therapy instituted at the time of EHEC diagnosis has shown to be somewhat protective against HUS development, but therapy remains non-specific and the factors that determine whether or not HUS develops are incompletely understood. Both HC and HUS are caused by Shiga toxins (Stx), which in most EHEC strains are encoded on prophages and released following prophage induction. Control of Stx production and release is encoded within the phage genome, but the factors that control Stx production and release in vivo are poorly characterized. We hypothesize that suppressing induction and/or phage functions during the diarrheic phase will suppress Stx in vivo and synergize with supportive therapy, to ameliorate or prevent the onset of HUS. The goal of this proposal is to identify phage functions that contribute to Stx production in vivo, and therefore could represent targets for Stx suppression in vivo. We will use the ; phage-based recombination system, recombineering, to generate mutations in specific phage genes, and evaluate the role of the encoded functions in Stx production and release both in vitro and in vivo. The two specific aims are: Aim 1: Determine the extent to which phage-mediated lysis, excision, circularization, and replication control Stx2 production and release. Aim 2: Determine which of the phage functions identified in aim 1 contribute to virulence in a well- characterized Shiga toxin-dependent animal model of disease. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic Escherichia coli (EHEC) are food-borne bacterial pathogens of humans that cause hemorrhagic colitis and occasionally hemolytic-uremic syndrome (HUS), a rapidly progressive often fatal form of renal failure that most often affects children. HUS is an uncommon complication of disease due to EHEC, but its importance is amplified by its severity, its tendency to affect young children, and the absence of any specific treatment or preventative measure. The goal of this project is to identify new targets for therapy to prevent or ameliorate HUS due to EHEC.